The accumulation of mitochondrial DNA (mtDNA) mutations in cells is strongly related to aging-associated diseases. Imaging of single-nucleotide variation (SNV) in mtDNA is crucial for understanding the heteroplasmy of mtDNAs that harbor pathogenic changes. Herein, we designed a CRISPR/Cas9-mediated proximity ligation assay (CasPLA) for direct visualization of the ND4 and ND5 genes in the mtDNAs of single cells. Taking advantage of the high specificity of CRISPR/Cas9, CasPLA can be used to image SNV in the ND4 gene at single-molecule resolution. Using CasPLA, we observed a mtDNA-transferring process between different cells through a tunneling nanotube, which may account for the spreading of mtDNA heteroplasmy. Moreover, we demonstrated that CasPLA strategy can be applied for imaging of single copy genomic loci ( KRAS gene) in the nuclear genome. Our results establish CasPLA as a tool to study SNV in situ in single cells for basic research and genetic diagnosis.